N-terminal amino acid sequence, immunohistochemical localization and tissue distribution of a plasma membrane protein (Prot17) of rat enterocytes

Prot17, a protein of the basolateral membrane of rat small intestine with a mol.wt. of 17 kDa, can be isolated using a previously described method (Schiechl 1988). It occurs in the membrane as an oligomer with a mol.wt. of 90 kDa. In the present study a polyclonal antibody specific for Prot17 was used to explore by immunohistochemical techniques the tissue distribution of Prot17 and its ultrastructural localization within the cells. Furthermore the amino acid sequence of the N-terminal part of this molecule up to position 17 could be analyzed. The results are summarized as follows: Prot17 is a membrane anchored protein. Its partial amino acid sequence suggests that it is neither identical nor related to other known proteins. Immunofluorescence studies revealed, that it occurs only in epithelial cells. It is mainly found in the absorptive and goblet cells of the intestine and the acinar cells of the pancreas. Smaller quantities are found also in the bile duct epithelium of the liver, in the proximal tubule cells of the kidney and in the cells of the respiratory epithelium. Ultrastructural localization of Prot17 was possible in the intestinal epithelium and pancreas acinar cells. In both cell types it was found in the basolateral and microvillous membrane. In pancreas, Prot17 was also detected in the membrane of the zymogen granules. In the absorptive cells of the intestine Prot17 was found in both the membrane and the contents of subluminal vesicles. Furthermore, in apical granules of secretory cells of the respiratory epithelium binding of Prot17 specific antibody was found in the granular content, the membrane being negative.     

Immunohistochemical localization of Na+-dependent glucose transporter in the rat digestive tract

Summary Glucose is actively absorbed in the intestine by the action of the Na⁺-dependent glucose transporter. Using an antibody against the rabbit intestinal Na⁺-dependent glucose transporter (SGLT1), we examined the localization of SGLT1 immunohistochemically along the rat digestive tract (oesophagus, stomach, duodenum, jejunum, ileum, colon and rectum). SGLT1 was detected in the small intestine (duodenum, jejunum and ileum), but not in the oesophagus, stomach, colon or rectum. SGLT1 was localized at the brush border of the absorptive epithelium cells in the small intestine. Electron microscopical examination showed that SGLT1 was localized at the apical plasma membrane of the absorptive epithelial cells. SGLT1 was not detected at the basolateral plasma membrane. Along the crypt-villus axis, all the absorptive epithelial cells in the villus were positive for SGLT1, whose amount increased from the bottom of the villus to its tip. On the other hand, cells in the crypts exhibited little or no staining for SGLT1. Goblet cells scattered throughout the intestinal epithelium were negative for SGLT1. These observations show that SGLT1 is specific to the apical plasma membrane of differentiated absorptive epithelial cells in the small intestine, and suggest that active uptake of glucose occurs mainly in the absorptive epithelial cells in the small intestine.     

Immunolocalization of potassium-chloride cotransporter polypeptides in rat exocrine glands

Potassium-chloride cotransporters (KCCs) encoded by at least four homologous genes are believed to contribute to cell volume regulation and transepithelial ion transport. We have studied KCC polypeptide expression and immunolocalization of KCCs in rat salivary glands and pancreas. Immunoblot analysis of submandibular, parotid, and pancreas plasma membrane fractions with immunospecific antibodies raised against mouse KCC1 revealed protein bands at ca 135 kDa and ca 150 kDa. Immunocytochemical analysis of fixed salivary and pancreas tissue revealed basolateral KCC1 distribution in rat parotid and pancreatic acinar cells, as well as in parotid, submandibular, and pancreatic duct cells. KCC1 or the polypeptide product(s) of one or more additional KCC genes was also expressed in the basolateral membranes of submandibular acinar cells. Both immunoblot and immunofluorescence signals were abolished in the presence of the peptide antigen. These results establish the presence in rat exocrine glands of KCC1 and likely other KCC polypeptides, and suggest a contribution of KCC polypeptides to transepithelial Cl(-) transport.     

Immunohistochemical characterization of a crypt cell-specific plasma membrane protein in rat small intestine epithelium using a monoclonal antibody

Proteins of the basolateral membrane (BLM) of small intestine epithelial cells of adult rats, in the MW ranges of 50-65 KD, 85-100 KD, and over 100 KD, were obtained as follows. After isolation of the BLM and subsequent SDS-PAGE and transblotting of the proteins on nitrocellulose sheets, the bands in these MW ranges were cut out of the nitrocellulose sheet and extracted. Balb/C mice were immunized with these protein fractions and a monoclonal antibody (MAb) was then produced. MAb SI/CC1 obtained via immunization with the 50-65 KD protein fraction shows specificity for the crypt epithelium of the small intestine. It can be used to characterize, by light and electron microscopic immunohistochemical methods, a crypt cell protein (SI/CC1-Ag) with a very specific localization. Fluorescence labeling shows that the SI/CC1-Ag can be found only in the epithelium of small intestine crypts (except for the granules in eosinophilic granulocytes). The epithelium of the colon, as well as the epithelia of other organs, could not be labeled. In the small intestine crypts, SI/CC1-Ag is found only in the Paneth cells located in the basal crypt section, and in the undifferentiated cells in the middle crypt section; it is lacking in the cells of the upper crypt section. Gold labeling shows that SI/CC1-Ag in the undifferentiated cells is localized exclusively in the basolateral PM domain. On the Paneth cells, the content of the secretory granules is labeled, along with the basolateral PM domain; the labeling sometimes present on their luminal part is probably due to passively absorbed secretion from these cells. The SI/CC1-Ag in the BLM of undifferentiated and Paneth cells is found only on Days 21-23 post partum, whereas the Paneth cell granules could be labeled as early as the Day 16 post partum. With immunodetection with SI/CC1, one band at about 55 KD is specifically labeled in the protein pattern of the isolated small intestine cell BLM. In the protein pattern of the isolated crypt cells two bands were labeled, again one at 55 KD and one at about 120 KD. These findings indicate that SI/CC1-Ag is a 55 KD protein that appears on Days 21-23 post partum in the BLM of undifferentiated cells and of Paneth cells.     

Cloning and immunolocalization of a rat pancreatic Na(+) bicarbonate cotransporter.

In the rat, pancreatic HCO(-)(3) secretion is believed to be mediated by duct cells with an apical Cl(-)/HCO(-)(3) exchanger acting in parallel with a cAMP-activated Cl(-) channel and protons being extruded through a basolateral Na(+)/H(+) exchanger. However, this may not be the only mechanism for HCO(-)(3) secretion by the rat pancreas. Recently, several members of electrogenic Na(+)/HCO(-)(3) cotransporters (NBC) have been cloned. Here we report the cloning of a NBC from rat pancreas (rpNBC). This rpNBC is 99% identical to the longer, more common form of NBC [pNBC; 1079 amino acids (aa); 122 kDa in human heart, pancreas, prostate, and a minor clone in kidney]. The longer NBC isoforms are identical to the rat and human kidney-specific forms (kNBC; 1035 aa; 116 kDa) at the approximately 980 C-terminal aa's and are unique (with different lengths) at the initial N-terminus. Using polyclonal antibodies to the common N- and C-termini of rat kidney NBC, a approximately 130-kDa protein band was labeled by immunoblotting of rat pancreas homogenate and was enriched in the plasma membrane fraction. Immunofluorescence and immunoperoxidase light microscopy of rat pancreatic tissue with both antibodies revealed basolateral labeling of acinar cells. Labeling of both apical and basolateral membranes was found in centroacinar cells, intra- and extralobular duct, and main duct cells. The specificity of the antibody labeling was confirmed by antibody preabsorption experiments with the fusion protein used for immunization. The data suggest that rpNBC likely plays a more important role in the transport of HCO(-)(3) by rat pancreatic acinar and duct cells than previously believed.     

Identification of a novel aquaporin, AQP12, expressed in pancreatic acinar cells

Members of the aquaporin (AQP) water channel family are widely distributed in various tissues and contribute to the water permeability of epithelial and endothelial cells. Currently 11 members of the AQP family (AQP0-10) have been reported in mammals. Here we report the identification of AQP12, which we found by performing a BLAST program search. Northern blot analysis revealed that AQP12 was specifically expressed in the pancreas. Further analysis by in situ hybridization and RT-PCR studies showed that AQP12 was selectively localized in the acinar cells of the pancreas. To analyze the cellular localization and function of AQP12, we expressed AQP12 in Xenopus oocytes and cultured mammalian cells. Immunocytochemistry revealed that AQP12 was not targeted to the plasma membrane. The selective localization of AQP12 in pancreatic acinar cells and possibly in the intracellular organelles suggests a role of AQP12 in digestive enzyme secretion such as maturation and exocytosis of secretory granules.     

In vivo localization of insulin binding to cells of the rat pancreas

The uptake of 125I-insulin by rat pancreas was studied in vivo. Following fixation and light microscope autoradiography, saturable uptake of 125I-insulin was quantitatively demonstrated on acinar and duct cells but not on blood vessels and islets of Langerhans. Electron microscopy revealed the localization of 125I-insulin to the basolateral cell membranes of acinar and duct cells.     

Cysteine string protein (CSP) is an insulin secretory granule-associated protein regulating beta-cell exocytosis.

Cysteine string proteins (CSPs) are novel synaptic vesicle-associated protein components characterized by an N-terminal J-domain and a central palmitoylated string of cysteine residues. The cellular localization and functional role of CSP was studied in pancreatic endocrine cells. In situ hybridization and RT-PCR analysis demonstrated CSP mRNA expression in insulin-producing cells. CSP1 mRNA was present in pancreatic islets; both CSP1 and CSP2 mRNAs were seen in insulin-secreting cell lines. Punctate CSP-like immunoreactivity (CSP-LI) was demonstrated in most islets of Langerhans cells, acinar cells and nerve fibers of the rat pancreas. Ultrastructural analysis showed CSP-LI in close association with membranes of secretory granules of cells in the endo- and exocrine pancreas. Subcellular fractionation of insulinoma cells showed CSP1 (34/36 kDa) in granular fractions; the membrane and cytosol fractions contained predominantly CSP2 (27 kDa). The fractions also contained proteins of 72 and 70 kDa, presumably CSP dimers. CSP1 overexpression in INS-1 cells or intracellular administration of CSP antibodies into mouse ob/ob beta-cells did not affect voltage-dependent Ca2+-channel activity. Amperometric measurements showed a significant decrease in insulin exocytosis in individual INS-1 cells after CSP1 overexpression. We conclude that CSP is associated with insulin secretory granules and that CSP participates in the molecular regulation of insulin exocytosis by mechanisms not involving changes in the activity of voltage-gated Ca2+-channels.     

Immunohistochemical localization of Na+-dependent glucose transporter in rat jejunum

Summary Glucose is actively absorbed via a Na⁺-dependent active glucose transporter (Na-GT) in the small intestine. We raised a polyclonal antibody against the peptide corresponding to amino acids 564–575 of rabbit intestinal Na-GT, and localized it immunohistochemically in the rat jejunum. By means of immunofluorescence staining, Na-GT was located at the brush border of the absorptive epithelial cells of the intestinal villi. Electron-microscopic examination showed that Na-GT was localized at the plasma membrane of the apical microvilli of these cells. Little Na-GT was found at the basolateral plasma membrane. Along the crypt-villus axis, all of the absorptive epithelial cells in the villus were positive for Na-GT. In addition to the brush border staining, the supranuclear positive staining, which was shown to be the Golgi apparatus by use of electron microscopy, was seen in cells located between the base to the middle of the villus. Cells in crypts exhibited little or no staining for Na-GT. Goblet cells scattered in the intestinal epithelium were negative for Na-GT staining. These observations show that Na-GT is specific to the apical plasma membrane of the absorptive epithelial cells, and that the onset of Na-GT synthesis may occur near the crypt-villus junction.     

Aminopeptidase N- and human blood group A-antigenicity along the digestive tract and associated glands in the rabbit

Summary The cellular localization of an aminopeptidase N homologous to the brush-border intestinal enzyme and that of human blood group A-substances were investigated using the immunofluorescence technique on thin frozen sections (200 nm) of the digestive tract and associated glands of A⁺ and A^– rabbits. Aminopeptidase N was found to be a common specific marker of both the apical region of plasma membrane of acinar cells in submaxillary and parotid glands and pancreas and the brush border of jejunum and colon absorbing cells. In hepatocytes, the enzyme was localized in the sinusoidal domains. Soluble A-substances were present in mucus secretory granules of intestinal goblet cells and those of stomach and gall bladder mucous cells. In contrast, the mucous acini of sublingual and submaxillary glands were devoid of A-antigenicity. The columnar cells of striated ducts of these glands exhibited A-antigenicity. Soluble A-substances were also found in zymogen granules of parotid and pancreas acinar cells and those of stomach chief cells. Moreover, in all cells secreting A-substances, and in the non-secreting absorbing intestinal cells, the glycoproteins of the plasma membrane bore A-determinants. Aminopeptidase N was one of the membrane-bound glycoproteins that bore A-determinants in cells that expressed A-antigenicity.     

Colocalization of ferroportin-1 with hephaestin on the basolateral membrane of human intestinal absorptive cells

An iron exporter ferroportin-1 (FPN-1) and a multi-copper oxidase hephaestin (Heph) are predicted to be expressed on the basolateral membrane of the enterocyte and involved in the processes of iron export across the basolateral membrane of the enterocyte. However, it is not clear where these proteins are exactly located in the intestinal absorptive cell. We examined cellular localization of FPN-1 and Heph in the intestinal absorptive cells using the fully differentiated Caco-2 cells. Confocal microscope study showed that FPN-1 and Heph are located on the basolateral membrane and they are associated with the transferrin receptor (TfR) in fully differentiated Caco-2 cells grown on microporous membrane inserts. However, Heph protein was not detected in the crypt cell-like proliferating Caco-2 cell. In stably transfected human intestinal absorptive cells expressing human FPN-1 modified by the addition of GFP at the C-terminus, we show that FPN-1-GFP is located on the basolateral membrane and it is associated with Heph suggesting the possibility that FPN-1 might associate and interact with Heph in the process of iron exit across the basolateral membrane of intestinal absorptive cell.     

Cellular and subcellular localization of annexin IV in rabbit intestinal epithelium, pancreas and liver.

The results of immunoblot analysis performed with a specific monoclonal antibody showed that the intestinal mucosa, pancreas and liver are privileged tissues for the expression of annexin IV. Immunofluorescence labelling of thin frozen sections of these tissues showed a strong concentration of annexin IV along the basolateral domain of the plasma membrane of intestinal absorbing cells, hepatocytes and pancreatic acinar cells, whereas in intestinal mucous secreting cells and centro acinar pancreatic cells, annexin IV was found to be present throughout the cytoplasm.     

Immunohistochemical localization of vascular endothelial growth factor in the globule leukocyte/mucosal mast cell of the rat respiratory and digestive tracts

 Vascular endothelial growth factor (VEGF) is a potent angiogenic mitogen that also increases vascular permeability. Immunohistochemical localization of VEGF in the respiratory and digestive tracts of healthy adult rats was investigated at light and electron microscopic levels using a specific antibody. The results revealed solitary cells with strong VEGF immunoreactivity scattered in the epithelium of the respiratory tract as well as in the lamina propria and epithelium of the intestine. From ultrastructural features of their large cytoplasmic granules, VEGF-positive cells in the respiratory tract were identified as globule leukocytes (GL). The immunoreactivity was localized exclusively in the cytoplasmic granules of GL. Most of the VEGF-positive cells in the small intestine were located in the lamina propria, whereas those in the large intestine were found more frequently in the epithelium than in the lamina propria. They showed the same morphological features as respiratory tract GL and were identified as mucosal mast cells (MMC). When examined in serial sections, GL/MMC in the respiratory and digestive tracts showed only weak reactivity to anti-histamine antibody. In contrast, connective tissue mast cells (CTMC), which were located in the submucosa of the digestive tract and in the connective tissues of the respiratory tract and other organs, were intensely immunopositive for histamine, whereas they showed no reactivity to anti-VEGF antibody. The specific occurrence of VEGF in GL/MMC suggests that this cell type is involved in paracrine regulation of the permeability of nearby microvessels, and that VEGF immunoreactivity can be used as a histochemical marker to distinguish GL/MMC from CTMC. Accepted: 28 July 1998     

Effect of colchicine on rat small intestinal absorptive cells. I Formation of basolateral microvillus borders

Treatment of rats with colchicine (0.5 mg/100 g of body weight) for more than 3 hr causes formation of microvillus borders along lateral and basal surfaces of absorptive cells in the small intestine. Morphologically, these strongly resemble the apical brush border inclusive of the terminal-web region. Formation of basolateral microvilli is restricted to mature absorptive cells. At 6 hr after administration of colchicine, 3.47% (+/- 1.94%) of the basolateral cell surfaces exhibit "implantation" of microvillus borders. The results show that colchicine induces formation of surface differentiations at lateral and basal surface regions that are restricted to the apical cell surface in controls. Redistribution of constituents of the plasma membrane from apical to basolateral membrane portions, as well as rearrangement in the organization of microfilaments can be considered to underlie formation of basolateral microvillus borders. From the antimicrotubular effect of colchicine it may be deduced that microtubules exert a regulative function in the formation of surface differentiations on absorptive cells of the small intestine and in the maintenance of the polarity of the cells.     

Expression and membrane localization of MCT isoforms along the length of the human intestine

Recent studies from our laboratory and others have demonstrated the involvement of monocarboxylate transporter (MCT)1 in the luminal uptake of short-chain fatty acids (SCFAs) in the human intestine. Functional studies from our laboratory previously demonstrated kinetically distinct SCFA transporters on the apical and basolateral membranes of human colonocytes. Although apical SCFA uptake is mediated by the MCT1 isoform, the molecular identity of the basolateral membrane SCFA transporter(s) and whether this transporter is encoded by another MCT isoform is not known. The present studies were designed to assess the expression and membrane localization of different MCT isoforms in human small intestine and colon. Immunoblotting was performed with the purified apical and basolateral membranes from human intestinal mucosa obtained from organ donor intestine. Immunohistochemistry studies were done on paraffin-embedded sections of human colonic biopsy samples. Immunoblotting studies detected a protein band of 39 kDa for MCT1, predominantly in the apical membranes. The relative abundance of MCT1 mRNA and protein increased along the length of the human intestine. MCT4 (54 kDa) and MCT5 (54 kDa) isoforms showed basolateral localization and were highly expressed in the distal colon. Immunohistochemical studies confirmed that human MCT1 antibody labeling was confined to the apical membranes, whereas MCT5 antibody staining was restricted to the basolateral membranes of the colonocytes. We speculate that distinct MCT isoforms may be involved in SCFA transport across the apical or basolateral membranes in polarized colonic epithelial cells. monocarboxylate transporter; short-chain fatty acids; absorption; short-chain fatty acid transport; mammalian colon     

Tissue injury alters the site of expression of hepatocyte growth factor activator inhibitor type 1 in bronchial epithelial cells

Hepatocyte growth factor activator inhibitor-1 (HAI-1) is a Kunitz-type transmembrane serine proteinase inhibitor that inhibits trypsin-like serine proteinases, such as hepatocyte growth factor activator, matriptase, hepsin and prostasin. HAI-1 is expressed in polarized epithelial cells, in which HAI-1 is mainly located on the basolateral membrane. In the present study, we analyzed the expression and distribution of HAI-1 in respiratory epithelium. We found that HAI-1 is expressed by the bronchial respiratory epithelium with basal or basolateral localization and also by the alveolar epithelium. Bronchial expression of HAI-1 was also confirmed using cultured human bronchial epithelial cells. The epithelial expression of HAI-1 was augmented in response to tissue injury such as cancer invasion and inflammation. Surprisingly, in the injured pulmonary tissue, HAI-1 showed distinct apical translocation in ciliated epithelial cells of the bronchiole. We suggest that, in addition to its basolateral surface localization, HAI-1 can transiently localize to the apical surface of respiratory ciliated epithelial cells under conditions of severe inflammation, possibly interacting with a specific cellular proteinase on the apical surface.     

Cellular Distribution and Subcellular Localization of mCLCA1/2 in Murine Gastrointestinal Epithelia

mCLCA1/2 are members of the CLCA protein family that are widely expressed in secretory epithelia, but their putative physiological role still awaits elucidation. mCLCA1/2 have 95% amino acid identity, but currently no specific antibody is available. We have generated a rabbit polyclonal antibody (pAb849) against aa 424–443 of mCLCA1/2. In HEK293 cells transfected with mCLCA1; pAb849 detected two specific protein bands at ∼125 kDa and 90 kDa, representing full-length precursor and N-terminal cleavage product, respectively. pAb849 also immunoprecipitated mCLCA1 and labeled the protein by immunostaining. But pAb849 crossreacted with mCLCA3/4/6 despite ≤80% amino acid identity of the antigenic epitope. We therefore investigated the cellular localization of mCLCA1/2 in epithelial tissues, which do not express mCLCA3/4/6 (salivary glands, pancreas, kidney) or express mCLCA3/6 with known localization (mucus cells of stomach and small intestine; villi of small intestine). mCLCA1/2 mRNA and protein expression were found in both parotid and submandibular gland, and immunohistochemistry revealed labeling in parotid acinar cells, in the luminal membrane of parotid duct cells, and in the duct cells of submandibular gland. In exocrine pancreas, mCLCA1/2 expression was restricted to acinar zymogen granule membranes, as assessed by immunoblotting, immunohistochemistry, and preembedding immunoperoxidase and immunogold electron microscopy. Moreover, mCLCA1/2 immunolabeling was present in luminal membranes of gastric parietal cells and small intestinal crypt enterocytes, whereas in the kidney, mCLCA1/2 protein was localized to proximal and distal tubules. The apical membrane localization and overall distribution pattern of mCLCA1/2 favor a transmembrane protein implicated in transepithelial ion transport and protein secretion. (J Histochem Cytochem 58:653–668, 2010)     

Immunohistochemical studies of the distribution of a basolateral-membrane protein in intestinal epithelial cells (GZ1-Ag) in rats using monoclonal antibodies

Summary The monoclonal antibody (mAb), GZ1, is specific for a 42-kilodalton (kD) protein (designated GZ1-Ag) present among the plasma membrane (PM) proteins of the absorptive cells of rat intestine. This protein only occurs in the basolateral PM and is absent from the microvillus membrane. GZ2 and GZ20 are two other mAbs that are also directed against GZ1-Ag but which specify other antigenic determinants of this protein than mAb GZ1. Used together, these three mAbs allow better characterization of GZ1-Ag and more precise investigation of its distribution and localization in various rat cells. We performed immunohistochemical labelling for GZ1-Ag at both the light-and electron-microscope levels and found that GZ1-Ag is extensively distributed in rat epithelial tissues. However, the amount of this protein present in epithelial tissue shows considerable variation. GZ1-Ag is not present in the secretory cells of terminal portions of most excretory glands or in cells of the endocrine glands and liver. The cells of kidney tubules, except for collecting tubules, also lack GZ1-Ag. Only small amounts of GZ1-Ag are present in the cells of the stratified squamous epithelium and transitional epithelium, the exception being superficial cells. High concentrations of GZ1-Ag occur in the excretory duct systems of glands and in the various kinds of epithelium present in the male and female genital tract. Our results also indicated that the GZ1-Ag in all of these cells has a very similar structure. In all cells, GZ1-Ag is localized in the PM, but it is present throughout the entire PM only in the cells of the stratified squamous epithelium and in the basal and intermediate cells of the transitional epithelium. In all epithelial cells bordering directly on the lumen, it is only present in the basolateral part of the PM, being absent from the luminal PM.     

Absorption of horseradish peroxidase by the small intestinal epithelium in postnatal developing rats.

After an intraluminal injection of horseradish peroxidase into the small intestine, the localization of peroxidase was studied in neonatal developing and adult rats by means of electron microscopy. Until around the 14th day of the neonatal period absorbed peroxidase granules in the duodenal and jejunal epithelium were abundant in the microvillous membrane, the apical tubulo-vacuolar system, and the Golgi apparatus, and on the lateral cell and basal membranes, and the luminal surfaces of the capillary cells. At the weaning period the tubulo-vacuolar system was absent in the duodenal and jejunal epithelial cells, and at that point absorbed peroxidase was observed in the same sites as in the adult rats: the microvillous membrane, the lateral cell and basal membranes, the Golgi apparatus, and the vesicles and vacuoles of the cytoplasm. During the suckling period, in the ileal epithelial cells exogenous peroxidase was found on the microvilli, in the tubulo-vacuolar system, in the supranuclear vacuole, in the Golgi apparatus, on the lateral cell and basal membranes, and also on the luminal surface of the endothelial cells of blood capillaries. When the tubulo-vacuolar system and the supranuclear vacuole were lost from the ileal cells at the weaning period, no exogenous peroxidase uptake was observed in the absorptive cell of the ileal epithelium.